Einleitung: Der Verdacht, dass Hunde mit einem Bandscheibenvorfall eine Prädisposition für die Entstehung einer Pankreatitis haben könnten, wurde in der Veterinärmedizin bereits in den frühen 1980er Jahren diskutiert. Trotz dieser bereits vor vielen Jahren erhobenen Vermutungen, wurde der Zusammenhang zwischen der Entstehung einer Pankreatitis und einem zeitgleich vorliegenden Bandscheibenvorfall auch im Hinblick auf mögliche Risikofaktoren wie dem Einfluss der Narkose oder dem Einsatz von Medikamenten (v. a. Glukokortikoide und nichtsteroidale Antiphlogistika) bisher nicht näher untersucht. Ziele der Untersuchungen: Im Rahmen der vorliegenden prospektiven Studie sollte untersucht werden, ob Bandscheibenvorfälle ein Risikofaktor für die Entstehung einer Pankreatitis beim Hund darstellen. Ferner sollte geklärt werden, ob die Narkose und die Gabe von Glukokortikoiden und/oder nichtsteroidalen Antiphlogistika zusätzlich das Risiko der Entstehung einer Pankreatitis bei Hunden mit einem Bandscheibenvorfall erhöhen. Material und Methoden: Insgesamt wurden 106 Hunde, bei denen aufgrund der klinischen Symptome der Verdacht einer Rückenmarksläsion bestand, an fünf aufeinander folgenden Tagen klinisch untersucht. Besonderes Augenmerk wurde hierbei auf Symptome gelegt, welche typischerweise bei Pankreatitiden zu beobachten sind (reduziertes Allgemeinbefinden, Schwäche, Anorexie, dolentes Abdomen, Vomitus, Regurgitieren, Diarrhoe, Fieber, Dehydratation). Ferner wurde am Tag 0 und Tag 4 der stationären Aufnahme die Konzentration der caninen pankreasspezifischen Lipase im Serum gemessen (Spec cPL und Snap cPL). Am Tag 0 wurde von dem Vorliegen einer Pankreatitis ausgegangen, wenn klinische Befunde im Sinne einer Pankreatitis sowie eine abnorm erhöhte Konzentration der caninen pankreasspezifischen Lipase im Serum (>400 μg/L) auffällig waren. Am Tag 4 erfolgte zudem eine sonographische Untersuchung des Abdomens. Somit basierte die Diagnosestellung einer Pankreatitis an diesem Tag auf dem Vorliegen von zwei der folgenden drei Kriterien: klinische Befunde im Sinne einer Pankreatitis, abnorm erhöhte Konzentration der caninen pankreasspezifischen Lipase im Serum, sonographische Hinweise für das Vorliegen einer Pankreatitis. Im Rahmen der statistischen Auswertung wurden zudem auch Patienten erfasst, welche in einem oder in mehreren der oben genannten Kriterien ein fragliches Ergebnis aufwiesen. Entsprechend ihrer neurologischen Ausfallserscheinungen sowie der Befunde im Rahmen der bildgebenden Diagnostik (Myelographie, Computertomographie, Kernspintomographie) wurden die Patienten in eine der folgenden drei Untersuchungsgruppen eingeteilt: 1. Hunde mit einem chirurgisch versorgten Bandscheibenvorfall (n = 71) 2. Hunde mit einem konservativ therapierten Bandscheibenvorfall (n = 20) und 3. Hunde mit einer akuten intramedullären Läsion (n = 15). Die statistische Auswerte erfolgte aufgrund der geringen Stichprobengrößen vorwiegend deskriptiv. Die Daten wurden mittels des Shapiro-Wilk-Tests auf Normalverteilung überprüft, die durchgeführten Gruppenvergleiche erfolgten unter Verwendung des Kruskal-Wallis und Mann-Whitney-U-Tests. Zudem wurden die betrachteten Merkmale mit dem Fisher Test und dem Chi-Quadrat-Test auf Unabhängigkeit überprüft. Das Signifikanzniveau wurde für alle Tests mit p < 0,05 festgelegt. Ergebnisse: Basierend auf den klinischen Symptomen und der Konzentration der caninen pankreasspezifischen Lipase im Serum konnte insgesamt am Tag 0 bei vier Hunden (3,8 %) eine Pankreatitis diagnostiziert werden. Am Tag 4 waren es, basierend auf den drei Kriterien, welche für die Diagnosestellung einer Pankreatitis herangezogen werden, insgesamt acht Patienten (7,5 %). Hunde mit einem Bandscheibenvorfall (chirurgisch beziehungsweise konservativ therapiert) wiesen am Tag 0 beziehungsweise Tag 4 in 4,3 % (n = 4) beziehungsweise 7,7 % (n = 7) der Fälle eine Pankreatitis auf. Aufgrund der geringen Häufigkeiten in den einzelnen Untersuchungsgruppen, war eine Berechnung eines signifikanten Unterschieds zwischen den Gruppen nicht möglich. Hinsichtlich einer möglichen Korrelation zwischen einer Narkose und der Entstehung einer Pankreatitis bei Hunden mit einer Rückenmarksläsion konnte kein signifikanter Zusammenhang festgestellt werden. Auch die Gabe von Glukokortikoiden und/oder nichtsteroidalen Antiphlogistika hatte hier keinen signifikanten Einfluss auf die Entstehung einer Pankreatitis. Schlussfolgerung: Vergleicht man die Ergebnisse der vorliegenden Studie mit der in der Literatur angegebenen Prävalenz für akute Pankreatitiden beim Hund (0,7-3,5 %), so kann geschlussfolgert werden, dass eine Rückenmarksläsion, insbesondere ein Bandscheibenvorfall, als Risikofaktor für die Entstehung einer akuten Pankreatitis beim Hund in Betracht gezogen werden muss. Demgegenüber erhöhen weder die Narkose noch die Gabe von Glukokortikoiden und/oder nichtsteroidalen Antiphlogistika zusätzlich das Risiko der Entstehung einer Pankreatitis bei Hunden mit einer Rückenmarksläsion. / Objective: The suspicion that dogs with intervertebral disc disease are at greater risk of developing pancreatitis is being discussed in veterinary medicine since the early 1980s. So far no study has been published examining the correlation between intervertebral disk disease and the development of pancreatitis in dogs, especially in combination with general anaesthesia and anti-inflammatory medication (glucocorticoids and/or nonsteroidal anti-inflammatory drugs). The aim of this study was therefore 1) to evaluate intervertebral disk disease as possible risk factor of pancreatitis and 2) to ascertain if general anaesthesia and the administration of glucocorticoids and/or nonsteroidal anti-inflammatory drugs further increase the risk of pancreatitis in dogs with intervertebral disk disease. Material and methods: One hundred and six dogs with symptoms associated with spinal cord injury were clinically examined over a period of five days. Special attention was payed to symptoms usually seen with pancreatitis such as anorexia, vomitus and abdominal pain. Furthermore the concentration of canine pancreatic lipase in the blood serum was measured with Spec cPL and Snap cPL at day 0 and day 4 after admission. At day 0 the diagnosis of pancreatitis was based on clinical symptoms associated with pancreatitis in combination with an increased concentration of canine pancreatic lipase in the blood serum (>400 μg/L). A sonography of the pancreas was performed at day 4 to evaluate the organ itself and the surrounding tissue for lesions associated with pancreatitis. Therefore the diagnosis of pancreatitis at day 4 was based on positive results in at least two of the three following criteria: symptoms associated with pancreatitis, elevation of the concentration of canine pancreatic lipase in the blood serum, sonographic changes of the pancreas parenchyma and the surrounding tissue associated with pancreatitis. For statistical analysis questionable results in one or more of these criteria were also documented. According to the neurologic symptoms and the findings of diagnostic imaging (myelography, computed tomography and magnetic resonance imaging), dogs were categorized in one of the following groups: 1. dogs with surgically treated intervertebral disk disease (n = 71), 2. dogs with medically treated intervertebral disk disease (n = 20), 3. dogs with an acute intramedullary lesion (n = 15). Due to the small sample size, statistics were primarily performed descriptively. Data were tested for normal distribution using the Shapiro-Wilk test. If Group comparisons were feasible, they were performed using the Kruskal-Wallis test and the Mann-Whitney-U test. Fisher test and the Chi-Square test were used to test for association between group affiliation and possible risk factors for the development of pancreatitis. A value of P < 0.5 was considered significant for all analysis. Results: Based on clinical symptoms and an elevated concentration of the canine pancreatic lipase (> 400μg/l) at day 0, four dogs (3.8 %) were diagnosed with pancreatitis. According to the clinical symptoms, the concentration of the canine pancreatic lipase and sonographic changes, a total number of eight dogs (7.5 %) were diagnosed with pancreatitis at day 4. Considering only the dogs with intervertebral disk disease (surgically and medically treated) 4.3 % (n = 4) and 7.7 % (n = 7) were diagnosed with pancreatitis at day 0 and day 4, respectively. Due to the small sample size, the calculation of significant differences between the three subgroups was not feasible. There was no significant correlation between general anaesthesia and the development of pancreatitis. Furthermore, the administration of glucocorticoids and/or nonsteroidal anti-inflammatory drugs is not significantly associated with the genesis of pancreatitis.

Chronic lower back pain is a major public health problem, with direct and indirect economic costs comparable to those of heart disease, depression and diabetes. In many cases this pain derives from degeneration of the intervertebral disc (IVD), a fibrous, avascular tissue that sits between the vertebrae in the spinal column. A novel treatment approach for this ‘discogenic’ pain is the injection of a hydrogel that hybridises in situ and restores the normal biomechanical function of the disc. While a number of promising materials are currently under development, existing approaches to removing degenerate material from the disc prior to injection are invasive and compromise the structural integrity of the disc. Mechanical fractionation of the tissue using acoustic cavitation generated by high intensity focussed ultrasound (HIFU) has the potential to be non-invasive, and to enhance the effectiveness of the procedure by preserving the outer regions of the disc. The primary goal of this thesis is to investigate the feasibility of this approach. The acoustic properties of the disc were first measured using a modified scanning acoustic microscope. The outer region of the disc, the annulus fibrosus (AF) was found to be highly attenuative compared to the central nucleus pulposus (NP). These measured properties were then used in a simplified two-dimensional model to simulate the shape of the acoustic pressure field within the disc. A configuration using two confocal spherically focussed 0.5 MHz single-element transducers was able to produce a tightly focused field suitable for use in the IVD. As preliminary experiments suggested that high pressure amplitudes were required to initiate cavitation inside the disc, the use of exogenous nuclei to lower this threshold was investigated. A novel class of solid sonosensitive nanoparticles (SNPs) suitable for use in the IVD were developed and characterised. These SNPs comprise a layer of hydrophobic silica particles deposited onto a polystyrene core, and are thought to trap small gas pockets in surface crevices. Coated particles were found to reduce the cavitation threshold significantly in both water and blood, from some 2.0 - 2.5 MPa at 1.067 MHz to below 1.0 MPa. The particles were also found to provide repeatable initiation of cavitation activity during prolonged or repeated exposures, and to exhibit good storage stability, suggesting that they they may be appropriate for use within the IVD. Finally, a combined therapy and monitoring system was designed, built and validated. The system comprised two confocal 0.5 MHz spherically focussed HIFU transducers with central openings, each co-axially aligned with either a single element passive cavitation detector or a 64-element array that could be used for both active and passive imaging. The system was found to be capable of initiating inertial cavitation in the disc at pressures as low as 2.5MPa in the presence of sonosensitive nanoparticles. Use of the array in active mode enables creation of a B-mode image that provides anatomical information on the boundaries of the IVD, whist the same array could be used for passive mapping of acoustic emissions arising fromthe HIFU focus during therapy. Two different exposure regimes were found to be capable of producing sizeable perforations within the NP without significantly damaging the AF, and preliminary investigations were carried out into themechanism of damage. The location and extent of cavitation as seen on passive maps acquired during treatment was found to coincide with the regions of NP fractionation. This confirms that passive acoustic mapping can provide the real-time treatment monitoring necessary to ensure both safety and efficacy of ultrasonic IVD fractionation. Prior to clinical application, a significant amount of further development is required to further validate non-invasive disc fractionation by HIFU and the subsequent steps for minimally invasive disc replacement using injectable hydrogels. The present work has nonetheless demonstrated for the first time that minimally invasive removal of degenerate disc tissue is feasible trough the combined use of sonosensitive nanoparticles and a relatively low-cost therapeutic ultrasound system that provides simultaneous anatomical imaging and real-time treatment monitoring by passive acoustic mapping.

Title: Human axial system: identification of connective tissues changes Objectives: The main objective of this thesis was to compile a review of techniques and methods currently used in identification of connective tissues changes. Methods: The method used in this thesis is a critical literature review - a study of research papers from available information sources accompanied by author's comments. Foreign sources are represented mostly by research papers accessible via electronic archives such as ScienceDirect, Pubmed, Springer, Wiley. Also other foreign publications were used. The theoretical part is focused on basic anatomy and physiology of the spine and states main methods of identification of connective tissues changes involved in this area. The main part describes particulars of researches dedicated to identification of functional and morphological characteristics of different spinal components. Results: In addition to classic methods of spinal research, the thesis introduces also new developing techniques and methods. Procedures used in current research are described; their advantages and limits are explained. Key words: spine, biomechanics, loading, intervertebral disc, method

Foi avaliada a participação dos corpos vertebrais e discos intervertebrais na lordose lombar e, a contribuição destes nas curvaturas lombares de diferentes magnitudes. Foram avaliadas as radiografias lombares em perfil de 350 adultos assintomáticos (143 homens e 207 mulheres, idade média 29 anos). Foram mensuradas a curvatura lombossacra (L1S1), a curvatura lombolombar (L1L5), a angulação de cada corpo vertebral e cada disco intervertebral por meio de uma variação do método de Cobb. A participação percentual dos corpos vertebrais e dos discos intervertebrais na curvatura lombossacra também foi determinada. Comparações entre os sexos e as faixas etárias foram realizadas. Os indivíduos foram divididos em três subgrupos populacionais, de acordo com a magnitude da lordose lombossacra, de modo a separar os indivíduos pertencentes aos extremos da curva de distribuição. Os componentes da curvatura lombar (corpos vertebrais e discos intervertebrais) foram comparados nestes três subgrupos. A medida da curvatura lombossacra no grupo inicial foi -60,9o (-33o a -89o). Os corpos vertebrais eram cifóticos em L1 (2,15o), tendiam ao neutro em L2 (-0,36o) e eram progressivamente lordóticos de L3 (-1,56o) a L5 (-9,23o). Os discos intervertebrais eram progressivamente lordóticos (variando de -4,99o em L1-L2 a -15,58o em L5-S1). Os corpos vertebrais e discos intervertebrais apresentaram participação progressivamente maior na curvatura lombossacra no sentido crânio-caudal. Os discos intervertebrais participaram com cerca de 80% da curvatura lombossacra, sendo que os elementos mais caudais (corpos vertebrais L4 e L5 e discos intervertebrais L4-L5 e L5-S1) corresponderam a mais de 65% da curvatura lombossacra. Os indivíduos mais velhos apresentaram medidas das curvaturas lombares maiores cerca de 4º em comparação aos indivíduos mais jovens, havendo diferença significante para as medidas dos corpos vertebrais L2 e L5 e o disco intervertebral L3-L4, sendo maiores as medidas nos indivíduos mais velhos. As medidas das curvaturas lombares e dos corpos vertebrais L2 e L4 apresentaram diferença estatisticamente significante entre os sexos, sendo as medidas maiores nos indivíduos do sexo feminino. A curvatura lombossacra apresentou média de -46,9° no subgrupo lordose menor; -61,59° no subgrupo lordose intermediária e; -74,13° no subgrupo lordose maior. A curvatura lombolombar apresentou média de -33,28° no subgrupo lordose menor; -45,34° no subgrupo lordose intermediária e; -56,96° no subgrupo lordose maior. Os corpos vertebrais e os discos intervertebrais apresentaram medidas absolutas menores no subgrupo lordose menor do que as dos subgrupos lordose intermediária e lordose maior, mas a participação dos discos intervertebrais na curvatura lombossacra no subgrupo lordose menor (88%) foi maior que nos subgrupos lordose intermediária (81%) e no subgrupo lordose maior (75%). Complementarmente, os corpos intervertebrais apresentaram maior participação nos subgrupos lordose maior e lordose intermediária. Individualmente, os corpos vertebrais apresentaram maior participação no subgrupo lordose maior, exceto pelo corpo vertebral L5 que apresentou maior participação no subgrupo lordose menor. A maior participação percentual dos discos intervertebrais no subgrupo lordose menor era devida à inclinação cifótica dos corpos vertebrais mais cefálicos (especialmente L1 e L2) no subgrupo lordose menor do que nos demais subgrupos, que, por um efeito compensatório, causava uma maior participação discal nas curvaturas menores. Os demais subgrupos apresentavam os corpos vertebrais cefálicos com inclinação muito mais lordótica do que o observado no subgrupo lordose menor. Concluímos que os discos intervertebrais são os principais responsáveis pela curvatura lombar e que a contribuição dos corpos vertebrais e discos intervertebrais na lordose lombar difere entre indivíduos com curvaturas de diferentes magnitudes. Apesar de ocorrer um aumento gradual do acunhamento lordótico do corpo e disco a cada nível vertebral conforme aumenta a medida da lordose, as vértebras mais cefálicas provocam uma diferença na contribuição percentual entre discos intervertebrais e corpos vertebrais nas curvaturas de tamanhos diferentes. / The vertebral bodies and intervertebral discs participation in lumbar lordosis and their contribution between lumbar curves of different size were studied. 350 lumbar spine radiographs of asymptomatic adults (143 men and 207 women, average age 29 years) were evaluated. Lumbosacral (L1S1) and lumbolumbar (L1L5) curves and the angular inclination of each vertebral boby and intervertebral disc were measured using a Cobb method variant. The percentile participation of each vertebral body and intervertebral disc in the lumbossacal curve was calculated. Sex and age were compared. The subjects were separated in tree subgroups, in acording to lumbosacral curve size. The compounds of lumbar curve (discs and vertebrae) were compared in these tree subgroups. The mean lumbosacral curve was ?60,9º (-33º to ?89º). L1 vertebral body was kyphotic (2,15º), L2 was neutral (-0,36º), and the other ones were progressively lordotic from L3 (-1,56º) to L5 (-9,23º). The intervertebral discs were progressively lordotic from L1-L2 (?4,99º) to L5-S1 (?15,58º). Both vertebrae and discs showed a progressive participation in cephalic-caudal direction. The participation of discs was about 80% of lumbosacral curve, and the caudal elements (L4, L5 vertebrae and L4-L5, L5-S1 discs) contributed far 65% of the curve. The older subjects presented lumbar curves larger than younger 4º average, with significant statistical difference to L2, L5 and L3-L4 measures, with older subjects presenting bigger angular values. There were statistical differences of lumbar curves, L2 and L4 measures between sexes, with females presenting bigger values. The lumbosacral curve presented average -46,9º in minor lordosis subgroup, -64,59º in intermediate lordosis sugbroup, and ?74,13º in major lordosis subgroup. The lumbolumbar curve presented average ?33,28º in minor lordosis subgroup, -45,34º in intermediate lordosis subgroup, and ?56,96º in major lordosis subgroup. The absolut values of vertebrae and discs angles were smaller in minor lordosis subgroup than in major lordosis subgroup, but the intervertebral discs participation of was bigger in minor lordosis subgroup (88%) than intermediate lordosis (81%) and major lordosis (75%) subgroups. Complementarely, the vertebrae had a bigger participation in intermediate and major lordosis subgroups. Individually, the vertebrae presented a larger participation in major lordosis subgroup, excepting L5 that presented bigger participation in minor lordosis subgroup. The discs presented larger participation in minor lordosis subgroup. That is consequence of a more kyphotic inclination of the cephalic vertebrae in minor lordosis subgroup than the other ones, causing a compensating effect, with a larger disc participation in the small curves. The intermediate and major lordosis subgroups had the cephalic vertebrae more lordotic than that of the minor lordosis subgroup. We concluded that the intervertebral discs are the main responsible for the lumbar curve angulation and that the contribution of vertebrae and discs in lumbar curves of different sizes is not equal. In spite of a gradual increase of lordotic wedging while lumbar curve increase, the cephalic vertebrae make the disc and vertebrae participation different between different magnitude lumbar curves.

Lower back disorders due to whole-body vibration (WBV) are the most common injuries reported by professional drivers. Such injuries often have long-term complications leading to significant personal and societal costs. An improved mathematical model of the whole human body would contribute to a better understanding of the mechanisms of lower back injury and be valuable in injury prevention research. Current biodynamic human models reported in the literature lack detailed information for predicting the non-linearity due to vibration amplitude of transmission of vibration from seat to a human. Therefore, one of the primary objectives of this research has been to develop and validate a detailed threedimensional biodynamic human model, with special attention given to the incorporation of active trunk muscles with non-linear stiffness properties. These muscles have been incorporated into an existing spine and neck model of a MADYMO 50th percentile male occupant model. A detailed multi-body human model has been developed, called MODEL ONE. This thesis shows that incorporating non-linear stiffness functions and energy dissipation using hysteresis or damping into a human model is appropriate for predicting non-linear biodynamic responses in arbitrary excitation functions. A major advantage of MODEL ONE compared to other multi-body models and lumped mass models is its ability to predict nonlinear seat-to-human transmissibility. However MADYMO 50th male occupant models use simplified geometry and rigid bodies to represent the lower lumbar spine. These simplified spinal models have no ability to simulate the internal stresses and deformations of soft tissues, even if these are the apparent cause of lower back pain (LBP). Therefore a detailed finite element human lower lumbar spine model - with appropriate material properties and capable of simulating internal stresses⎯is necessary, in order to better understand spinal injuries under WBV. A three-dimensional finite element model of a lower lumbar spine motion segment - called MODEL TWO - has thus been developed for the present study. MODEL TWO comprises a detailed geometric description of vertebrae, nucleus pulposus, endplates, and intervertebral discs. The intervertebral discs lump together the annulus fibrosus, ground substance and ligaments. The vertebrae have been assumed to be rigid. The material properties of the intervertebral discs of MODEL TWO were obtained from test matrices and from various parameter data reported in the literature. MODEL TWO has been validated against cadaveric experiments reported in the literature. The mechanical behaviour and stress distribution within the MODEL TWO intervertebral disc agree reasonably well with the cadaveric experiments. MODEL TWO was integrated into MODEL ONE to form a new human model, called MODEL THREE, which was subsequently dynamically validated against volunteers� responses to WBV reported in the literature. MODEL THREE, as presented in this thesis, consists of a multi-body human model with detailed representation of a finite element (FE) lower lumbar spine. As far as the author is aware, MODEL THREE is the first model with detailed representation of a FE lower lumbar spine to successfully demonstrate that it is capable of simulating the stress profile of the entire intervertebral disc and endplate region due to WBV. The simulated results revealed abnormal stress concentrations in both the posterior and xviii the posterolateral annulus. The stresses increased most in the posterolateral intervertebral discs region during WBV, suggesting a possible mechanism for disc mechanical overload leading to fatigue fracture and degeneration. The results from MODEL THREE are promising and lead to a more comprehensive understanding of the behaviour of the intervertebral disc under WBV. MODEL THREE has also provided a good foundation for the development of a bio-fidelity human model. However, implementation of currently unavailable and/or inadequate in vitro and in vivo experimental studies is needed to further validate and develop MODEL THREE. A better understanding of injury mechanisms and the clinical significance of LBP will ultimately be arrived at using a combination of analytical models with in vitro and in vivo experimental data.

Degeneration of the lumbar intervertebral disc, a condition widely implicated in the cause of low back pain among adult humans, is typically characterised by progressive biochemical and structural changes to the extracellular matrix. Comprehensive descriptions of the structural and functional inter-relationships within the extracellular matrix are therefore critical to understanding the degenerative process and developing effective treatments. In the anulus fibrosus, this matrix has a complex, hierarchical architecture comprised of collagens, proteoglycans, and elastic fibres. Elastic fibres are critical constituents of dynamic biological structures that functionally require elasticity and resilience. Studies to date of elastic fibre network structure in the anulus fibrosus have been qualitative and limited in scope. Additionally, there is poor understanding of the structural and functional associations between elastic fibres and other matrix constituents such as collagen, and, critically, there have been no studies directly examining the nature and magnitude of the contribution made by elastic fibres to anulus fibrosus mechanical behaviour. In this thesis, multiple experimental studies are described that specifically examine each of these areas. Novel imaging techniques were developed and combined with histochemistry and light microscopy to facilitate the visualisation of elastic fibres at a level of detail not previously achieved. Examination of elastic fibre network structure revealed architectural differences between the intralamellar and interlamellar regions, suggesting that elastic fibres perform functional roles at distinct levels of the anulus fibrosus structural hierarchy. The density of elastic fibres within lamellae was found to be significantly higher in the lamellae of the posterolateral region of the anulus than the anterolateral, and significantly higher in the outer regions than the inner, suggesting it may be commensurate with the magnitude of the tensile strains experienced by each region of the disc in bending and torsion. The nature of the structure-function associations between elastic fibres and collagen was then examined with respect to the reported structural mechanisms of collagen matrix tensile deformation. Histological assessment of collagen crimp morphology in specimens from which elastic fibres had been enzymatically removed revealed no observable differences when compared with controls, suggesting that any contribution made by elastic fibres to maintaining crimp is minimal. Elastic fibres in anulus fibrosus specimens subjected to radial tensile deformations exhibited complex patterns of re-arrangement, suggesting that they maintain cross-collagen fibre connectivity. Elastic fibres were also observed to maintain physical connections between consecutive lamellae undergoing relative separation. Finally, the nature and magnitude of the contribution made by elastic fibres to anulus fibrosus mechanical properties at the tissue level was investigated using a combination of biochemically verified enzymatic treatments and biomechanical tests. Targeted degradation of elastic fibres resulted in a significant reduction in both the initial modulus and the ultimate modulus, and a significant increase in the extensibility, of radially oriented anulus fibrosus specimens. Separate treatments and mechanical tests were used to account for any changes attributable to non-specific degradation of glycosaminoglycans. These results suggest that elastic fibres enhance the mechanical integrity of the anulus fibrosus extracellular matrix in the direction perpendicular to the plane containing the collagen fibres. In summary, the results of the studies presented in this thesis provide important new insights into the structure and function of the anulus fibrosus elastic fibre network, and highlight its potential importance as a contributing or ameliorating factor in the progression of the structural and mechanical changes associated with intervertebral disc degeneration. Additionally, these results establish an improved framework for the development of more accurate analytical and finite element models to describe disc behaviour. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1317006 / Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2008

Low back pain represents a significant concern in the United States, with 70% of individuals experiencing symptoms at some point in their lifetime. Although the specific cause of low back pain remains unclear, symptoms have been strongly associated with degeneration of the intervertebral disc. Insufficient nutritional supply to the disc is believed to be a major mechanism for tissue degeneration. Understanding nutrients' transport in intervertebral disc is crucial to elucidate the mechanisms of disc degeneration, and to develop strategies for tissue repair (in vivo), and tissue engineering (in vitro). Transport in intervertebral disc is complex and involves a series of electromechanical, chemical and biological coupled events. Despite of the large amount of studies performed in the past, transport phenomena in the disc are still poorly understood. This is partly due to the limited number of available experimental techniques for investigating transport properties, and the paucity of theoretical or numerical methods for systematically predicting the mechanisms of solute transport in intervertebral disc. In this dissertation, a theoretical and experimental approach was taken in order to investigate the mechanisms of solute transport and binding interactions in intervertebral disc. New imaging techniques were developed for the experimental determination of diffusive and binding parameters in biological tissues. The techniques are based on the principle of fluorescence recovery after photobleaching, and allow the determination of the anisotropic diffusion tensor, and the rates of binding and unbinding of a solute to the extracellular matrix of a biological tissue. When applied to the characterization of transport properties of intervertebral disc, these methods allowed the establishment of a relationship between solute anisotropic and inhomogeneous diffusivity and the unique morphology of human lumbar annulus fibrosus. A mixture theory for charged hydrated soft tissues was presented as a framework for theoretical investigations on solute transport and binding interactions in cartilaginous tissues. Based on this theoretical framework and on experimental observations, a finite element model was developed to predict solute diffusive-convective-reactive transport in cartilaginous tissues. The numerical model was applied to simulate the effect of mechanical loading on solute transport and binding interactions in cartilage explants and intervertebral disc.

Crosslinking soft tissue has become more common in tissue engineering applications, and recent studies have demonstrated that soft tissue mechanical behavior can be directly altered through crosslinking, but increased understanding of how crosslinking affects intervertebral disc mechanical behavior is needed. In vitro testing of bovine disc and motion segments was used to characterize several important aspects of disc behavior in response to crosslinking after both soaking and injection treatment. The first study was a comparison of different crosslinkers to determine the effect on tensile properties of disc tissue. Circumferential specimens were taken from bovine annulus and then soak treated with an optimized crosslinking formulation or sham solution. A non-contacting laser micrometer was used to measure cross sectional area, after which tension testing until failure was performed to determine yield strain, yield stress, ultimate stress, peak modulus, and resilience. The crosslinkers were observed to produce different changes in the properties, with the measured properties generally increasing. The second study used bilateral annular injections to simulate a clinically relevant delivery method. The dose response of the motion segment’s neutral zone stability metrics against injection concentration was mapped. Concentrations of 20 mM and less had no significant effects on the stability metrics. 40mM demonstrated a change in neutral zone stiffness, while at least 80mM was required to significantly affect neutral zone length. Thus, meaningful changes in joint neutral zone stability were demonstrated using clinically relevant injection and chemical formulations. The third study used combinations of biochemical and accelerated mechanical cyclic loading to degrade gelatin and annulus fibrosus specimens with and without genipin treatment. Genipin crosslinking attenuated changes during cyclic loading to specimen geometry and compliance relative to control samples. Full recovery of genipin treated samples appeared to be hampered, at least partially from continued crosslinking during the accelerated testing. The fourth study tested the effect of genipin crosslinking to resist interlamellar shearing of the annulus lamella. Using a recently reported test method that shears adjacent lamella, crosslinked specimens were noted to have significantly higher yield force, peak force, and resilience compared to sham treated controls, supporting the hypothesis that crosslinking would increase the load bearing ability of the interface.

<p>Intervertebral disc (IVD) disorders are a major contributor to disability and health costs. Disc disorders and resulting pain may be preceded by changes which first occur in the nucleus pulposus (NP) region of the IVD, where significant alterations in tissue cellularity, composition, and structure begin early in human life and continue with increasing age and degeneration. These changes coincide with the loss of a distinct cell population, notochordally-derived immature NP cells, which may play a key role in the generation and maintenance of this tissue. These cells reside in a gelatinous, highly-hydrated extracellular matrix (ECM) environment and exhibit in situ cell-matrix and cell-cell interactions which are quite distinct from cells in other regions of the disc or in other cartilagenous, including expression of laminin cell-matrix receptors and cell-associated laminin proteins. The ECM environment is known to be a key regulator of cellular behaviors, with ECM ligands and elasticity modulating cell adhesion, organization, differentiation, and phenotype. The primary motivating hypothesis of this thesis is that the unique ECM environment of the NP plays a key role in modulating immature NP cell behaviors, and that laminin ligands, in combination with ECM elasticity, modulate immature NP cell behaviors including adhesion, organization, and phenotype.</p><p>To investigate this hypothesis, flow cytometric analyses were performed to examine IVD cell integrin receptor expression over time in culture, including assessment of key laminin-binding integrin subunits. The roles of specific integrin receptors modulating NP cell adhesion to ECM proteins were identified in studies utilizing function-blocking antibodies. NP cell adhesion, spreading, and relative cell adhesion strength was assessed on various ECM constituents, including specific isoforms of laminin. Additionally, studies were performed to examine the roles of ECM ligand and substrate stiffness in modulating NP cellular organization, utilizing polyacrylamide gel substrates with tunable mechanical properties and functionalized with different ECM ligands. Finally, the role of ECM environment was examined on one key measure of NP cell function, proteoglycan production, over time in culture.</p><p>NP cells isolated from immature NP tissues were found to express high levels of the laminin-binding integrin subunit alpha 6 ex situ and maintain this expression over time in culture. Function blocking studies revealed this receptor to be a key regulator of NP cell adhesion to laminin, in contrast to cells from the adjacent AF region, which did not express this receptor nor adhere to laminin. Cell adhesion studies demonstrated that NP cells preferentially interact with two laminin isoforms, LM-511 and LM-332, in comparison to other ECM proteins, with enhanced cell attachment, spreading, and adhesion strength on surfaces coated with these ligands. These findings correspond with laminin isoform and receptor expression patterns identified in immature NP tissues. Additionally, NP cell-cell interactions were found to be modulated by both ECM ligand and substrate stiffness, with soft, laminin-functionalized substrates promoting self-assembly of NP cells into cell clusters with morphologies similar to those identified in immature NP tissues. Finally, culture of immature NP cells on soft, laminin-rich substrates was found to promote a key measure of NP cell function, proteoglycan synthesis.</p><p>The studies presented here demonstrate that immature NP cells interact uniquely with laminin extracellular matrix proteins, and that laminin ligands and matrix elasticity are two key regulators of NP cell organization and phenotype in the IVD. These findings suggest that alterations in one or both of these factors during IVD aging or degeneration may contribute to the differentiation or loss of this unique cell population. Additionally, these results indicate that soft, laminin-functionalized biomaterials may be appropriate for in vitro culture and expansion of immature NP cells, as well as for use in NP tissue engineering strategies.</p> / Dissertation

<p>Intervertebral disc (IVD) disorders can cause pain and disability for affected individuals. A subset of IVD disorders are thought to originate in the nucleus pulposus (NP) region of the IVD, due to alterations in tissue structure and composition with age and injury. Cells of the NP undergo a phenotypic and behavioral shift with age, changes that are thought to disrupt tissue homeostasis and lead to disc degeneration. There is significant interest in developing biomaterials and strategies to revert adult degenerate NP cells to healthy, young NP cell phenotypes with increased biosynthesis and cell clustering. Studies demonstrate that healthy porcine NP cells interact with laminin proteins through specific integrin subunits on soft substrates in a process that regulates cell morphology and biosynthesis. The central hypothesis of this dissertation is that the engagement of cell-surface adhesion receptors, using laminin-mimetic peptides on a controlled stiffness material, can revert adult degenerate NP cellular phenotype and behaviors to their healthy, biosynthetically active form.</p><p>In the first part of this dissertation, integrin subunits used by adult degenerate human NP cells to attach to laminin were revealed using flow cytometric analyses, function blocking antibodies, and immunohistochemistry. Secondly, NP cell recognition peptides were identified by screening laminin-mimetic peptides for cell attachment. Finally, human NP cells were cultured on peptide functionalized polyacrylamide gels to examine the effect of ligand and substrate stiffness in regulating adult human NP cell phenotype and biosynthesis. </p><p>Findings reveal that adult human NP cells express and utilize integrin subunits α3, α5, and β1 to attach to laminins, in contrast to integrin α6β1 found previously for healthy porcine NP cells. This difference between current and previous findings likely arises from aging-associated difference in NP cells between human and porcine tissues. Select laminin-mimetic peptides, chosen from the literature and informed by NP cell integrin expression, were found to promote significant NP cell attachment in a concentration dependent manner. Finally, a subset of laminin mimetic peptides were found to promote a young NP cell phenotype by increasing cell clustering on soft (0.3 kPa) and stiff (14 kPa) substrates as well as increasing proteoglycan synthesis on soft substrates. </p><p>The studies presented in this dissertation demonstrate that adult degenerate human NP cells attach to laminin proteins in an integrin dependent manner. Furthermore, laminin-mimetic peptides are able to mediate human NP cell attachment at levels comparable to full-length laminin, increase cell clustering when presented on soft and stiff substrates, and can increase NP cell biosynthesis when presented on soft substrates. Utilizing laminin-mimetic peptides may allow for the design of biomaterials that promote a healthy young NP phenotype for a variety of disc therapies.</p> / Dissertation